Methods and compositions for assessing germline risk of cancer

Abstract

Heritable mutations in the BRCA1 and BRCA2 and other genes in the DNA double-strand break (DSB) repair pathway increase risk of breast, ovarian and other cancers. In response to DNA breaks, the proteins encoded by these genes bind to each other and are transported into the nucleus to form nuclear foci and initiate homologous recombination. Flow cytometry-based functional variant analyses (FVAs) were developed to determine whether variants in BRCA1 or other DSB repair genes disrupted the binding of BRCA1 to its protein partners, the phosphorylation of p53 or the transport of the BRCA1 complex to the nucleus in response to DNA damage. Each of these assays distinguished high-risk BRCA1 mutations from low-risk BRCA1 controls. Mutations in other DSB repair pathway genes produced molecular phenocopies with these assays. FVA assays may represent an adjunct to sequencing for categorizing VUS or may represent a stand-alone measure for assessing breast cancer risk.

What is claimed is: 
     
       1. A method comprising:
 obtaining circulating white blood cells from a subject; treating the white blood cells with a DNA damaging agent; performing a flow cytometry based functional variant analysis (FVA); 
 measuring in the treated cells BRCA2 nuclear localization; comparing a measured functional activity with at least one control value obtained from control white blood cells treated with the DNA damaging agent and having a wild type BRCA2 gene; and categorizing variants of the BRCA2 gene in the subject as functional, having loss of function or having a gain of function, based on the comparing step. 
 
     
     
       2. The method of  claim 1 , wherein said at least one control value is measured from normal white blood cells or white blood cells having a normal DNA DSB repair pathway. 
     
     
       3. The method of  claim 1 , wherein said at least one control value is measured from white blood cells with a defective DNA DSB pathway and cancer risks. 
     
     
       4. The method of  claim 1 , wherein said at least one control value is established at an earlier time. 
     
     
       5. The method of  claim 1 , wherein said at least one control value is established in parallel to the measurement of the functional activity of the DSB repair pathway in the white blood cells from said subject. 
     
     
       6. The method of  claim 1 , wherein said white blood cells are selected from the group consisting of B cells, lymphoblastoid cells and total white blood cells. 
     
     
       7. The method of  claim 1 , where the white blood cells are cultured prior to treatment with a DNA damaging agent. 
     
     
       8. The method of  claim 1 , wherein the DNA damaging agent is selected from radiation, a chemical compound, or a combination thereof. 
     
     
       9. The method of  claim 8 , wherein said radiation is UV, x-ray or gamma-ray. 
     
     
       10. The method of  claim 9 , wherein said compound is selected from the group consisting of Mitomycin C (MMC), 1,3-butadiene diepoxide (DEB), Bleomycin(Bleo), or a combination thereof. 
     
     
       11. The method of  claim 1 , wherein BRCA2 nuclear localization is measured in an assay using one DNA damaging agent. 
     
     
       12. The method of  claim 1 , wherein BRCA2 nuclear localization is measured in multiple assays each using a different DNA damaging agent. 
     
     
       13. The method of  claim 1 , wherein BRCA2 nuclear localization is measured using a DCW (digital cell Western) nuclear localization assay format. 
     
     
       14. The method of  1 , wherein the at least one functional activity is measured by a flow cytometer. 
     
     
       15. The method of  1 , wherein the white blood cell is a cultured cell. 
     
     
       16. The method of  claim 13 , wherein the DCW nuclear localization assay comprises
 fixing the treated cells, 
 subjecting the treated cells to cell lysis conditions that causes lysis of a predetermined percentage of cells, 
 collecting the nuclei and unlysed cells by centrifugation, 
 resuspending the collected nuclei and unlysed cells in a staining buffer comprising an optically active agent-conjugated anti-BRCA2 antibody and an optically active agent-conjugated antibody specific for a protein localized intracellularly and outside of the nucleus, 
 measuring the amount of BRCA2 protein localized to the nuclei and the total amount of BRCA2 protein in the cells, and 
 determining the ratio of the amount of BRCA2 protein localized to the nuclei versus the total amount of BRCA2 protein expressed in the cells as basis of determining said functional activity of BRCA2.